Linalool Dehydratase-Isomerase, a Bifunctional Enzyme in the Anaerobic Degradation of Monoterpenes

Abstract

Castellaniella (ex Alcaligenes) defragrans strain 65Phen mineralizes monoterpenes in the absence of oxygen. Soluble cell extracts anaerobically catalyzed the isomerization of geraniol to linalool and the dehydration of linalool to myrcene. The linalool dehydratase was present in cells grown on monoterpenes, but not if grown on acetate. We purified the novel enzyme ϳ1800-fold to complete homogeneity. The native enzyme had a molecular mass of 160 kDa. Denaturing gel electrophoresis revealed one single protein band with a molecular mass of 40 kDa, which indicated a homotetramer as native conformation. The aerobically purified enzyme was anaerobically activated in the presence of 2 mM DTT. The linalool dehydratase catalyzed in vitro two reactions in both directions depending on the thermodynamic driving forces: a water secession from the tertiary alcohol linalool to the corresponding acyclic monoterpene myrcene and an isomerization of the primary allylalcohol geraniol in its stereoisomer linalool. The specific activities (V max ) were 140 nanokatals mg ؊1 for the linalool dehydratase and 410 nanokatals mg ؊1 for the geraniol isomerase, with apparent K m values of 750 M and 500 M, respectively. The corresponding open reading frame was identified and revealed a precursor protein with a signal peptide for a periplasmatic location. The amino acid sequence did not affiliate with any described enzymes. We suggest naming the enzyme linalool dehydratase-isomerase according to its bifunctionality and placing it as a member of a new protein family within the hydrolyases (EC 4.2

Geraniol and Geranial Dehydrogenases Induced in Anaerobic Monoterpene Degradation by Castellaniella defragrans

“…A number of other GeDHs from plant sources (25,43,49) and the insect pest Carpoglyphus lactis (37) are involved in geraniol synthesis and therefore may not have evolved a higher affinity for geraniol than enzymes in degradation pathways. The transformation of ␤-myrcene to geraniol by the linalool dehydratase isomerase is thermodynamically unfavorable and results in a steady state at low geraniol concentrations (7,35), thus requiring a high-affinity GeDH for an efficient metabolic flux. Furthermore, geraniol was found to inhibit monoterpene metabolism at aqueous concentrations above 5 M in the two-liquid-phase system with geraniol in the organic carrier phase.…”

“…Biomass production yielded approximately 40 g (wet weight) cells grown on 15 mM limonene, ␣-phellandrene, or 100 mM acetate and 100 mM nitrate in a 10l fermentor (24). For large-scale, heterologous production of proteins, Escherichia coli BL21 Star(DE3) (Invitrogen, Darmstadt, Germany) carrying the overexpression plasmid was grown in terrific broth medium (7).…”

“…Geraniol exudes a sweet, rose-like scent and is commercially synthesized in amounts of 4,000 Mg year Ϫ1 (3). In C. defragrans, the linalool dehydratase-isomerase catalyzes the hydration of the acyclic ␤-myrcene to (S)-(ϩ)-linalool as well as the isomerization to geraniol (7,35). The formation of geranic acid was observed in vivo and in vitro (24), indicating the presence of dehydrogenases catalyzing the oxidation of the allyl alcohol geraniol to geranic acid, most probably via geranial.…”

“…Geraniol (3,7-dimethyltrans-2,6-octadien-1-ol) is a C 10 homologue of 3-methyl-2-buten-1-ol and is known to be an intermediate in the anaerobic degradation of ␤-myrcene by Castellaniella defragrans (7). This betaproteobacterium, originally named Alcaligenes defragrans (29), was isolated with various monoterpenes, natural unsaturated hydrocarbons (C 10 H 16 ) that can be simply differentiated by their acyclic, monocyclic, or bicyclic structure ( Fig.…”

Bacterial Degradation of tert-Amyl Alcohol Proceeds via Hemiterpene 2-Methyl-3-Buten-2-ol by Employing the Tertiary Alcohol Desaturase Function of the Rieske Nonheme Mononuclear Iron Oxygenase MdpJ

“…allylic rearrangement previously shown for the interconversion of the monoterpenes linalool and geraniol (13). Recently, the enzyme responsible for the rearrangement in the bacterium Castellaniella defragrans strain 65Phen was identified (6). Besides isomerization, this so-called linalool dehydratase-isomerase also catalyzes the dehydration of linalool to myrcene, very similar to the allylic rearrangement and dehydration of unsaturated tertiary alcohols catalyzed by plant monoterpene cyclases (48).…”